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Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
09:40

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Published on: September 28, 2018

Expression, purification, and inhibition of human RET tyrosine kinase

Luca Mologni1, Elisa Sala, Barbara Riva

  • 1Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy. luca.mologni@unimib.it

Protein Expression and Purification
|April 2, 2005
PubMed

Insights

Researchers developed a method to purify the RET tyrosine kinase domain for drug screening. This led to the identification of novel RET inhibitors for treating RET-driven thyroid cancers.

Area of Science:

  • Oncology
  • Biochemistry
  • Molecular Biology

Background:

  • Tyrosine kinases, including RET, are critical in cancer development.
  • Dysregulated RET kinase activity drives a significant portion of thyroid carcinomas.
  • Targeting RET offers a promising therapeutic strategy for these cancers.

Purpose of the Study:

  • To develop a method for expressing and purifying the recombinant RET kinase domain.
  • To establish a screening assay for identifying potential RET kinase inhibitors.
  • To facilitate the development of novel therapeutics for RET-driven malignancies.

Main Methods:

  • Purification of His-tagged RET kinase domain from Sf9 insect cells using a two-step chromatographic protocol.
  • Characterization of the purified recombinant RET kinase domain.
  • Development of a rapid ELISA-based kinase assay using a validated RET-specific peptide substrate.

Main Results:

  • Successfully expressed and purified active recombinant RET kinase domain.
  • Identified and validated a specific peptide substrate for RET kinase.
  • Developed and utilized a high-throughput ELISA assay to screen for inhibitors.
  • Discovered novel inhibitors targeting RET kinase activity.

Conclusions:

  • The developed recombinant RET kinase and assay are effective tools for inhibitor screening.
  • This work provides a foundation for developing targeted therapies against RET-driven thyroid cancers.
  • Novel RET inhibitors identified hold potential for future clinical applications.

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